Identification of Autism genes that regulate synaptic Nrx/Nlg signaling complexes

鉴定调节突触 Nrx/Nlg 信号复合物的自闭症基因

• 批准号: 8082598
• 负责人: Craig C Garner
• 金额: $ 23.11万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-08 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8082598
• 关键词: Adhesions; Adult; Affect; Applications Grants; Asperger Syndrome; Autistic Disorder; Behavioral; Biological Assay; Biotinylation; Brain region; Cadherins; Caring; Cell Adhesion Molecules; Characteristics; Child; Communication; Complex; Coupled; DLG4 gene; Data; Defect; Deletion Mutation; Diagnosis; Down-Regulation; Drug Delivery Systems; Equilibrium; Etiology; Excitatory Synapse; Genes; Genetic; Glutamates; Goals; Heterogeneity; Hippocampus (Brain); Image; Individual; Inhibitory Synapse; Label; Lead; Link; Literature; Mediating; Molecular; Monitor; Mus; Mutate; Mutation; N-Methyl-D-Aspartate Receptors; Neurodevelopmental Disorder; Neurons; Patients; Pervasive Development Disorder; Pharmacological Treatment; Pharmacotherapy; Phenotype; Plastics; Population; Prevalence; Probability; Productivity; Property; Proteins; Publishing; RNA Interference; Resolution; Screening procedure; Services; Signal Pathway; Signal Transduction; Small Interfering RNA; Social Behavior; Special Education; Specific qualifier value; Stereotyped Behavior; Symptoms; Synapses; Synapsins; Synaptic Vesicles; Synaptic plasticity; System; Technology; Testing; Therapeutic; autism spectrum disorder; base; cognitive function; contactin; cost; design; economic cost; experience; expression vector; gene function; high throughput screening; loss of function; mouse model; postsynaptic; presynaptic; public health relevance; small molecule libraries; synaptic function; vector; vocalization

DESCRIPTION (provided by applicant): Autism is a neurodevelopmental disorder characterized by abnormal social behavior, communication deficits, and repetitive or stereotyped behaviors. Cumulative prevalence literature suggests that approximately 1 in 1000 children are diagnosed with Autism, and as many as 1 in 150 are diagnosed with one of the Autism Spectrum Disorders (ASDs), including Asperger's Syndrome and PDD-NOS (pervasive developmental disorder not otherwise specified). Economic costs associated with ASDs are estimated at $35 billion/year, including special education services and treatments to reduce symptoms. These estimates do not even factor in the costs associated with lost productivity and specialized care for Autistic individuals once they reach adulthood. A key to developing therapeutic strategies to effectively treat ASDs is a fundamental understanding of the cellular and molecular mechanisms that underlie them. The goal of this grant application is to design an imaging-based screening assay in order to assess whether a group of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Our approach will not only define key molecular components of the signaling pathway(s) involved in ASDs, but also create a platform for screening small molecule libraries in order to identify potential pharmacotherapies for ASDs. Specifically, we will test the hypothesis that many of the genes mutated in ASD patients function to regulate the formation of complexes between two key synaptic proteins, the transsynaptic cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. We will first establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. Next, we will evaluate whether at least a subset of ASD-associated genes regulate the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs against known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, higher-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway, and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes. PUBLIC HEALTH RELEVANCE: The goal of this grant application is to design an imaging-based screening assay in order to assess whether subsets of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Specifically, we will test the hypothesis that a set of the genes mutated in ASD patients function to regulate the formation of transsynaptic complexes between two key synaptic proteins, the cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. In Aim 1, we will establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes in dissociated hippocampal cultures. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. In Aim 2, we will evaluate whether ASD-associated genes regulates the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs for the known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, high-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes.

描述（由申请人提供）：自闭症是一种神经发育障碍，其特征是社会行为异常，沟通缺陷以及重复或刻板印象的行为。累积患病率文献表明，大约有1000名儿童被诊断出患有自闭症，其中150人中有1000名儿童被诊断出患有一种自闭症谱系障碍（ASDS）之一，包括阿斯伯格综合征和PDD-NOS（未指定的普遍性发育障碍）。与ASD相关的经济成本估计为每年350亿美元，包括特殊教育服务和减少症状的治疗。这些估计甚至不会考虑与自闭症患者成年后的生产率失去和专业护理相关的成本。开发有效治疗ASD的治疗策略的关键是对它们构成的细胞和分子机制的基本理解。该赠款应用的目的是设计基于成像的筛选测定法，以评估一组与自闭症谱系障碍（ASDS）相关的基因是否位于共同的信号通路中。我们的方法不仅将定义ASD中涉及的信号传导途径的关键分子组件，而且还将创建一个筛选小分子库的平台，以识别ASDS的潜在药物疗法。具体而言，我们将测试以下假设：ASD患者中突变的许多基因在两个关键突触蛋白之间的形成，即跨突触细胞粘附分子神经毒素和神经素的形成，从而介导了兴奋性Glutamatamearmatialgic Synapses的成熟，功能和可塑性。我们将首先建立一个基于成像的测定法，以检测和量化透射性神经素/神经素（NRX/NLG）复合物的水平。 Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation.接下来，我们将评估至少与ASD相关基因的一个子集调节突触NRX/NLG复合物的形成。具体而言，我们将针对已知的ASD相关基因产物创建简短的干扰（SI）RNA，并执行中等通量筛选以评估这些分子的下调是否影响NRX/NLG复合物的形成。一旦到位，该测定方法将适应于siRNA和小分子文库的自动化，高通量的筛选，从而可以鉴定基于NRX/NLG的信号途径的其他分子成分，以及潜在的药物靶标，以使这些GENES中ASD患者的认知功能正常化。 公共卫生相关性：该赠款应用的目的是设计基于成像的筛选测定法，以评估与自闭症谱系障碍（ASDS）相关的基因子集（ASDS）是否位于共同的信号传导途径中。具体而言，我们将检验以下假设：ASD患者中突变的一组基因在两个关键突触蛋白之间的透射性突触复合物的形成，即细胞粘附分子神经毒素和神经素，从而介导了兴奋性Glutameartameartameartamataragic Synapses的成熟，功能和可塑性。在AIM 1中，我们将建立一个基于成像的测定法，以检测和量化分离的海马培养物中经突触神经素/神经素（NRX/NLG）复合物的水平。 Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation.在AIM 2中，我们将评估与ASD相关的基因是否调节突触NRX/NLG复合物的形成。具体而言，我们将为已知的ASD相关基因产物创建简短的干扰（SI）RNA，并执行中等通量筛选以评估这些分子的下调是否影响NRX/NLG复合物的形成。一旦到位，该测定方法将适应于siRNA和小分子文库的自动化高通量筛选，从而可以鉴定基于NRX/NLG的其他分子成分，基于NRX/NLG的信号途径和潜在药物靶标的潜在药物靶标在这些基因中携带的ASD患者的认知功能正常化。